Osteoarthritis is characterized by progressive enzymatic destruction of type II collagen and aggrecan, which are the two major components of cartilage matrix. Type II collagen is essential for cartilage tensile strength and its degradation causes progression of osteoarthritis.
Aggrecan is composed of a core protein of approximately 2400 amino acids. The molecule consists of several structural and functional domains (Falnnery et al., Matrix Biology 16, 1998, 507–511). Three domains are defined on the N-terminal side: (1) the G1, (2) the interglobular, and (3) the G2 domain. The aggrecan C-terminal side comprises two glycosaminoglycan rich domains. As shown in the aggrecan representation below, the G1 domain is separated from a second globular domain, G2, by about 150 amino acids, known as the interglobular domain. From the G2 domain to the C-terminus there is a long extended region consisting of two glycosaminoglycan-rich domains. The first is rich in keratan sulfate, whereas that which follows is rich in chondroitin sulfate.

The G1 domain of aggrecan binds to long hyaluronic acid polymers, thereby forming multi molecular aggregates that effectively immobilize aggrecan within the collagen fibrillar meshwork. The glycosaminoglycan domains provide osmotic pressure, which enables cartilage to resist compression.
Current osteoarthritis therapies (e.g., non-steroidal anti-inflamatories or NSAIDs) have limited symptomatic benefit and have only modest, if any, effects on slowing cartilage destruction in osteoarthritic joints. NSAIDs, such as, acetaminophen, act by inhibiting the synthesis of cytokines, such as, prostaglandins that cause pain, and swelling. Thus, NSAIDs do not directly prevent cartilage destruction, whereas inhibitors of cartilage degrading enzymes will block cartilage collagen and aggrecan degradation thereby blocking or slowing the progression of osteoarthritis. Thus, inhibition of the enzymes should have a more direct and specific effect on cartilage breakdown than cytokine inhibition.
The loss of aggrecan contributes to the progression of osteoarthritis. In osteoarthritis and rheumatoid arthritis, aggrecan is one of the first cartilage matrix components to undergo measurable loss (Mankin et al., J Bone Joint Surg. 52A, 424–434 (1970)).
In human arthritis, aggrecan degradation is associated with amino acid cleavage within the interglobular domain, at either the Asn341-Phe342 or the Glu373-Ala374 site. In vitro studies have demonstrated that the aggrecan Asn341-Phe342 bond can be cleaved by several collagenases including collagenase-1 and collagenase-3 (Fonsang et al., FEBS Lett. 380: 17–20, 1996a), however, digestion of aggrecan with a number of these purified proteases has not resulted in cleavage at the Glu373-Ala374 site (Fonsang et al., J. Biol. Chem. 267, 19470–19074, (1992); Flannery et al J. Biol. Chem. 267, 1008–1014 (1992)).
Recently, an enzyme that demonstrated aggrecanase proteolytic activity—i.e., cleaves the Glu373-Ala374, but not the Asn341-Phe342 site of aggrecan—has been identified (Arner et al., PCT publication WO 99/05291; Arner et al., J. Bio. Chem. 274 (10) 6594–6601 (1999); and Tortorella et al., Science 284, 1664–1666 (1999)). The enzyme was designated aggrecan degrading metalloprotease (ADMP) or ADAMTS-4.
WO 99/05291 teaches that the zymogen form of the isolated and purified ADMP consist of a propeptide domain containing a furin cleavage site, a metalloprotease domain, an a disintegrin-like domain, and a thrombospondin homologous domain (i.e., region of the molecule containing one or more thrombospondin type 1 (TSP1) repeats).
ADAMTS-4 is classified within the a disintegrin and metalloproteinase (ADAM or reprolysin) subfamily of the metazincins (Rawlings et al., Methods in Enzymology 248, 183–228 (1995) and Stocker et al., Protein Science 4, 823–840 (1996)). ADAMs represent a new family of genes that show a significant sequence similarity to snake venom metalloproteinase and disintegrin (Hite et al., Biochemistry 31, 6203–6211 (1992); Wolfberg et al., J. Cell Bio. 131, 275–278 (1995)).
Some ADAMS cause the release of inflammatory cytokines and the levels of these harmful ADAMs are often increased in joint disease. For example, ADAM-17—also known as tumor necrosis factor-alpha converting enzyme (TACE)—is responsible for cleavage of cell bound tumor necrosis factor-alpha (TNF-α). TNF-α is involved in many auto-immune diseases (W. Friers, FEBS letters 285, 199 (1991)). There are two forms of TNF-α, a type II membrane protein of relative molecular mass 26 kD and a soluble 17 kD form generated from the cell bound TNF-αby specific TACE proteolytic cleavage. The 17 kD form of TNF-α is released by the cell and is associated with the deleterious side effects of TNF-α. Thus, inhibitors of TACE prevent the formation of soluble TNF-α and so obviate its toxic side effects.
On the other hand, there are instances where compounds that inhibit matrix degrading enzymes-such as, MMP-13 and aggrecanase—but do not have a strong TACE inhibitory action, are preferred.
Other ADAMs include ADAMTS-1 (Kuno et al., J. Biol. Chem. 272, 556–562 (1997) and Tang et al., FEBS Letters 445, 223, 1999), and ADAMs 10, 12, and 15 (Wu et al., Biochem. Biophys. Res. Comm. 235, 437–442 (1997)).
Collagen destruction by the MMP subfamily of the zinc metalloendopeptidases is characteristic of some joint diseases, such as, osteoarthritis. The MMP subfamily contains seventeen identified members (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-18, MMP-19, MMP-20). The MMPs are known to regulate the turn-over of extracellular matrix proteins and as such play important roles in normal physiological processes, such as, reproduction, development, and differentiation. But the MMPs are also expressed in many pathological situations in which abnormal connective tissue turnover is occurring.
Three matrix metalloproteinases that degrade type II collagen are known, MMP-1, MMP-8, and MMP-13, referred to herein as collagenase-1, -2, and -3, respectively.
Collagenase-1 (MMP-1) is expressed in a wide variety of connective tissues throughout the body (e.g., skin, cartilage, gingiva, meniscus, tendon, and ligament) (Mitchell et al., J. Clin. Invest. 97, 761–768 (1996) and Wolfe et al., Arthritis Rheum. 36, 1540–1547 (1993)).
Collagenase-2 (MMP-8) is expressed primarily by neutrophils, but levels of MMP-8 mRNA and protein are present in human cartilage. It has been suggested that this enzyme may participate in aggrecan degradation (Chubinskaya et al., Lab. Invest. 74, 232–240 (1993) and Cole et al., J. Biol. Chem. 271, 11023–11026 (1996)).
Collagenase-3 (MMP-13) (Freije et al., J. Biol. Chem. 269, 16766–16773 (1994) is found almost exclusively in cartilage. This enzyme has been shown to significantly degrade type II collagen and, in addition, increased amounts are present in human osteoarthritic cartilage (Mitchell et al., J. Clin. Invest. 97, 761–768 (1996)).
Matrix metalloproteinase and reprolysin inhibitors are well known in the literature. Specifically, European Patent Publication 606,046, published Jul. 13, 1994, refers to certain heterocyclic MMP inhibitors. U.S. Pat. No. 5,861,510, issued Jan. 19, 1999, refers to cyclic arylsulfonylamino hydroxamic acids that are useful as MMP inhibitors. PCT Publication WO 98/34918, published Aug. 13, 1998, refers to heterocyclic hydroxamic acids including certain dialkyl substituted compounds that are useful as MMP inhibitors. PCT publications WO 96/27583 and WO 98/07697, published Mar. 7, 1996 and Feb. 26, 1998, respectively, refer to arylsulfonyl hydroxamic acids. PCT publication WO 98/03516, published Jan. 29, 1998 refers to phosphinates with MMP activity. PCT publication 98/33768, published Aug. 6, 1998, refers to N-unsubstituted arylsulfonylamino hydroxamic acids. PCT Publication WO 98/08825, published Mar. 5, 1998, refers to certain MMP inhibitors. Each of the above referenced publications and applications is hereby incorporated by reference in its entirety.
Non-selective collagenase inhibitors, i.e., inhibitors of a broad range of collagenases, are known to block collagen destruction in vivo (Nixon et al., Int. J. Tiss. React 13, 237–243 (1991); Mitchell et al., Annals. New York Acad. Sci. 732, 395–397 (1994); and Mort et al., Matrix 13, 95–102 (1993)). See also, PCT publications WO 96/33172 and WO 96/27583 which teach hydroxamic acids that broadly inhibit MMPs and WO 98/58925 which teaches barbiturate type MMP inhibitors.
Selective Matrix metalloproteinase and reprolysin inhibitors are disclosed in EP 935963, published Aug. 18, 1999, and U.S. Non-Provisional Patent Application “TACE Inhibitors”, filed Aug. 12, 1999, which refers to certain heterocyclic hydroxamic acid compounds with differential selectivity for MMP-13, MMP-1, TACE, and aggrecanase (both of which are incorporated by reference herein). See also U.S. Non-Provisional Patent Application “Pyrimidine 2,4,6-Trione Metalloprotease inhibitors”, filed Aug. 12, 1999 (incorporated herein by reference).
Although non-selective collagenase inhibitors are potential therapeutic agents, they can cause systemic connective tissue toxicity. For example, an inhibitor of both collagenase-3 and collagenase-1, revealed significant dose-related connective tissue side effects (Proceedings of ASCO, 15, 490 (1996)). Such connective tissue toxicity significantly limits the therapeutic utility of non-selective MMP inhibitors. It has been proposed that the toxicity of non-selective collagenase inhibitors results from suppression of normal connective tissue collagen turnover by collagenase-1. Collagenase inhibitors without collagenase-1 activity should therefore, have no or reduced connective tissue toxicity.
Diseases in which high potency inhibition of aggrecanase should provide therapeutic benefit include: osteoarthritis, joint injury, reactive arthritis, acute pyrophosphate arthritis, psoriatic arthritis, and rheumatoid arthritis. Of course, high potency inhibiting compounds are desired because lower doses can be effective. Furthermore, since it is recognized that varied combinations and concentrations of pathological enzymes are expressed in different joint diseases, compounds that inhibit several of the inflammation related proteases in addition to aggrecanase, are desirable.